Regulators of G Protein Signaling 6 and 7

Abstract

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein ␥ subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein ␤ subunits, and certain biochemical properties of RGS6/␤5 and RGS7/␤5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the G␤5 subunit (but not G␤s 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/␤5 in that they act as GTPase-activating proteins specifically toward G␣ o . Unlike conventional G ␤␥ complexes, RGS6/␤5 and RGS7/␤5 do not form heterotrimeric complexes with either G␣ o -GDP or G␣ q -GDP. Neither RGS6/␤5 nor RGS7/␤5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-␤1 or -␤2. However, the RGS/␤5 complexes inhibited ␤ 1 ␥ 2 -mediated activation of phospholipase C-␤2. RGS/␤5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G i and G o by binding to phospholipase C and stimulating the GTPase activity of G␣ o .Initial studies of proteins that belong to the regulators of G protein signaling (RGS) 1 family demonstrated that they are GTPase-activating proteins (GAPs) that accelerate hydrolysis of GTP bound to the ␣ subunits of certain heterotrimeric G proteins (1-3). As such, RGS proteins can function as negative regulators of G protein-mediated signal transduction by speeding deactivation of the active form of G ␣ subunits, thereby promoting formation of inactive G protein heterotrimers (G␣ GDP ␤␥). It is now clear that some proteins that contain an RGS domain have more complex functions. For example, p115 RhoGEF, which contains an RGS domain near its amino terminus, is an effector for G protein action, catalyzing guanine nucleotide exchange on the monomeric G protein Rho more efficiently when stimulated by G␣ 13 -GTP. The capacity of G␣ 13 to activate p115 RhoGEF is dependent on the RGS domain of p115, which also accelerates hydrolysis of GTP by G␣ 13 (4, 5). p115 RhoGEF thus resembles phospholipase C␤, a well characterized effector for G protein action that also deactivates its regulator (G␣ q ) by acting as a GAP (6 -8).A subfamily of RGS proteins has been identified in which each member possesses so-called DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein ␥ subunit-like) domains in addition to an RGS domain (9, 10). Members of this group include mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11), a Drosophila RGS protein (dRGS7), and EGL-10, an RGS protein found in Caenorhabditis elegans (10 -12) Functionally, the GGL domain was shown to specify binding of RGS11 or a fragment of RGS7 to the G protein ␤5 subunit (10). It has also been shown that both RGS7 and RGS9 can be isolated from brain and retina as a complex with G␤5 (13-15) 2 and that the distribution of mRNA for the GGL-containing RG...

Noncatalytic Domains of RGS9-1·Gβ5L Play a Decisive Role in Establishing Its Substrate Specificity

“…The idea that noncatalytic domains of RGS proteins may contribute to establishing the specificity in the RGS-G protein interactions is supported by several recent observations (5)(6)(7). Understanding the molecular mechanisms conferring this specificity has been complicated by many reports that RGS proteins and especially their catalytic domains can stimulate the GTPase activity of a broad spectrum of G protein ␣-subunits in vitro.…”

“…5 for additional examples). In contrast, some RGS proteins, like RGS4, seem to be rather nonspecific in interacting with their G␣ partners (5,26).…”

“…It has been shown that the catalytic domain of RGS7 stimulates the GTPase activities of G␣ i and G␣ o with equal efficiencies (26,30) whereas the full-length RGS7⅐G␤5 complex stimulates the GTPase activity of only G␣ o (5,30). Thus, the primary role of the noncatalytic domains of RGS9-1⅐G␤5L in determining its substrate specificity is likely to reflect a general principle in RGS protein functioning.…”

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

“…Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2,9,15). Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16)(17)(18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26).…”

Mechanisms Governing Subcellular Localization and Function of Human RGS2

“…This suggests that the regulatory selectivity observed in cells (18) is achieved by interaction with other signaling molecules. For example, the N terminus of RGS4 confers receptor-selective regulation of G qcoupled responses (19,20); the PDZ domain of RGS12 binds peptides from the C termini of the interleukin-8-RB receptor (21); the GGL domains of RGS6, RGS7, RGS9, and RGS11 selectively bind G␤ 5 (22)(23)(24)(25)(26)(27)(28)(29); and a Dbl-like domain in p115…”